The specificity of connections formed between proprioceptive sensory neurons and motor neurons in the developing spinal cord is dependent on a series of tightly controlled cellular interactions that culminate in the recognition of specific motor neuron dendrites by incoming sensory afferents. Emerging evidence indicates that transcription factors expressed by functional subsets of sensory and motor neurons direct the development of selective sensory-motor connections. One important unresolved issue is the identity of the relevant targets of the transcription factors that control motor and sensory axon growth and connectivity. The main goal of this proposal is to define the link between the transcriptional regulation of spinal motor and sensory neuron identity and the key cell surface recognition molecules that control axonal growth and target specificity in this neural circuit. Three specific aspects of motor and sensory neuron development will be addressed. Motor neurons located within lateral motor column project their axons differentially along the dorsoventral axis of the limb, under the control of LIM homeodomain transcription factors. We will use mouse genetic methods to explore the possibility that LIM homeodomain proteins direct motor axon guidance by regulating the expression of Eph kinases and ephrins on motor axons and limb mesenchymal cells. ETS class transcription factors regulate the trajectory and terminal axonal branching of both motor and sensory neurons, and genetic studies have revealed that ETS genes regulate semaphorin expression in motor neurons. We will use mouse genetic approaches to map the distribution, and analyze the function, of specific semaphorins and their major receptors, the plexins, in the differentiation of motor and sensory neurons, focusing on the function of these genes in sensory axonal growth and connectivity. One difficulty in identifying relevant targets of LIM homeodomain and ETS genes has been the limitation in number of primary neurons that can be obtained from mouse embryos. To overcome this problem, we will take advantage of the ability to generate motor neurons in essentially unlimited numbers from mouse ES cells, and the finding that ES cell-derived motor neurons lack expression of many of the LIM homeodomain and ETS proteins that regulate motor axonal growth and connectivity. ES cell derived motor neurons will therefore be used as a cellular system for DNA-based microarray screens to define and study the function of additional target genes that are activated or repressed by these transcription factors.